Azadirachta indica A. Juss Ameliorates Memory Deficits and Reduces Anxiety-Like Behavior by Modulating Cholinergic Neurotransmission in an Animal Model of Depression: In Silico and In Vivo Studies
,
Abstract
Exposure to prolonged and severe stress can lead to negative effects on learning and memory, increased anxiety, reduced motivation, disturbed cholinergic activity, and hippocampal and prefrontal cortical neuronal damage. On the other hand, drugs from natural origin have a beneficial effect on neuronal structure and functions. Azadirachta indica (neem), belonging to the Meliaceae family, has been reported to exhibit beneficial effects in wound healing, diabetes management, and antibacterial properties. This study aimed to assess the impact of Azadirachta indica on chronic immobilization stress-induced memory impairment in rats. Chronic immobilization stress was induced in rats for 2 hours/day over 10 days. Following this, Azadirachta indica was administered at doses of 200, 400, and 600 mg/kg for 14 days. Twenty-four hours after the treatment, behavioral tests, including the novel object recognition test (NORT), T-maze, and elevated plus maze (EPM), were conducted to evaluate memory and anxiety-like behaviors. Acetylcholinesterase (AChE) activity was measured in the frontal cortex, hippocampus, and septum. Additionally, molecular docking studies were performed using Molegro Virtual Docker (MVD-2013, 6.0) to analyze the interaction of 19 active chemical constituents from aqueous neem extracts with various targets, including AChE, brain-derived neurotrophic factor (BDNF), N-methyl-D-aspartate(NMDA) receptors, and anti-cortisol Fab in complex with corticosterone. Azadirachta indica treatment significantly enhanced learning and memory in chronically stressed rats, as evidenced by improved performance in NORT and T-maze tests, along with reduced anxiety-like behavior in the EPM test. Treatment also restored AChE activity in the stressed animals. Molecular docking studies indicated that the active constituents of neem extract showed high docking scores to AChE, BDNF, NMDA receptors, and anti-cortisol Fab, correlating with the experimental findings. Azadirachta indica exhibited neuroprotective and cholinergic transmission modulation properties, which may underlie its memory-enhancing effects in chronically stressed rats. Treatment enhanced memory in NORT and T-maze test (p<0.001). Also, anxiety behavior was reduced in the EPM (p<0.001). The correlation between the in vitro experimental data and the in silico molecular docking results suggests that neem’s active compounds could be potential candidates for improving memory and managing stress-induced cognitive impairments.
Schneiderman N, Ironson G, Siegel SD. Stress and Health: psychological, behavioral, and biological determinants. Annu Rev Clin Psychol 2005;1:607-628.
Selye H. The evolution of the stress concept. Stress and cardiovascular disease. Am J Cardiol 1970;26:289-299.
Bhagya V, Christofer T, Shankaranarayana Rao BS. Neuroprotective effect of Celastruspaniculatus on chronic stress-induced cognitive impairment. Indian J Pharmacol 2016;48: 687-693.
Bhagya V, Srikumar BN, Veena J, Shankaranarayana Rao BS. Short‐term exposure to enriched environment rescues chronic stress‐induced impaired hippocampal synaptic plasticity, anxiety, and memory deficits. J Neurosci Res 2017;95:1602-1610.
Shilpa BM, Bhagya V, Harish G, SrinivasBharath MM, Shankaranarayana Rao BS. Environmental enrichment ameliorates chronic immobilisation stress-induced spatial learning deficits and restores the expression of BDNF, VEGF, GFAP and glucocorticoid receptors. Prog Neuropsychopharmacol Biol Psychiatry 2017;76:88-100.
Shilpa BM, Bhagya V, Shankaranarayana Rao BS. Escitalopram treatment meliorates chronic immobilization stress induced depressive behavior and cognitive deficits by modulating BDNF expression in the hippocampus. J Appl Pharmaceutical Sci 2024;14:170-182.
Uno H, Tarara R, Else JG, Suleman MA, Sapolsky RM. Hippocampal damage associated with prolonged and fatal stress in primates. J Neurosci 1989;9:1705-1711.
Krishnan V, Nestler EJ. Animal models of depression: molecular perspectives. Curr Top Behav Neurosci 2011;7:121-147.
Mizoguchi K, Ishige A, Aburada M, Tabira T. Chronic stress attenuates glucocorticoid negative feedback: involvement of the prefrontal cortex and hippocampus. Neuroscience 2003;119:887-897.
Lupien SJ, McEwen BS, Gunnar MR, Heim C. Effects of stress throughout the lifespan on the brain, behaviour and cognition. Nat Rev Neurosci 2009;10:434-445.
Magariños AM, Verdugo JM, McEwen BS. Chronic stress alters synaptic terminal structure in hippocampus. Proc Natl Acad Sci U S A. 1997;94:14002-14008.
Adamec R, Hebert M, Blundell J, Mervis RF. Dendritic morphology of amygdala and hippocampal neurons in more and less predator stress responsive rats and more and less spontaneously anxious handled controls. Behav Brain Res 2012;226:133-146.
Kienzle-Horn S. Herbal medicines for neurological diseases. Curr Opin Investig Drugs 2002;3: 763-767.
Carlini EA. Plants and the central nervous system. Pharmacol Biochem Behav 2003;75:501-512.
Khan R, Krishnakumar A, Paulose CS. Decreased glutamate receptor binding and NMDA R1 gene expression in hippocampus of pilocarpine-induced epileptic rats: neuroprotective role of Bacopamonnieri extract. Epilepsy Behav 2008;12:54-60.
Kobayashi MS, Han D, Packer L. Antioxidants and herbal extracts protect HT-4 neuronal cells against glutamate-induced cytotoxicity. Free Radic Res 2000;32:115-124.
Abhijit S, Subramanyam MV, Devi SA. Grape seed proanthocyanidin and swimming exercise protects against cognitive decline: a study on M1 acetylcholine receptors in aging male rat brain. Neurochem Res 2017;42:3573-86.
Konar A, Gautam A, Thakur MK. Bacopamonniera (CDRI-08) upregulates the expression of neuronal and glial plasticity markers in the brain of scopolamine induced amnesic mice. Evid Based Complement Alternat Med 2015;2015:837012.
Alzohairy MA. Therapeutics role of Azadirachtaindica (neem) and their active constituents in diseases prevention and treatment. Evid Based Complement Alternat Med 2016;2016:7382506.
Bedri S, Khalil EA, Khalid SA, Alzohairy MA, Mohieldein A, et al. Azadirachtaindica ethanolic extract protects neurons from apoptosis and mitigates brain swelling in experimental cerebral malaria. Malar J 2013;12:298.
Yanpallewar S, Rai S, Kumar M, Chauhan S, Acharya SB. Neuroprotective effect of Azadirachtaindica on cerebral post-ischemic reperfusion and hypoperfusion in rats. Life Sci 2005; 76:1325-1338.
Vaibhav K, Shrivastava P, Khan A, Javed H, Tabassum R, et al. Azadirachtaindica mitigates behavioral impairments, oxidative damage, histological alterations and apoptosis in focal cerebral ischemia reperfusion model of rats. Neurol Sci 2013;34:1321-1330.
Ennaceur A, Meliani K. A new one-trial test for neurobiological studies of memory in rats III. Spatial vs. non-spatial working memory. Behav Brain Res 1992;51:83-92.
Ennaceur A. One-trial object recognition in rats and mice: Methodological and theoretical issues. Behav Brain Res 2010;215:244-254.
Graeff FG, Netto CF, Zangrossi H Jr. The elevated T-maze as an experimental model of anxiety. Neurosci Biobehav Rev 1998;23:237-246.
Golli NE, Dallagi Y, Rahali D, Rejeb I, Fazaa SE. Neurobehavioral assessment following e-cigarette refill liquid exposure in adult rats. Toxicol Mech Methods 2016;26:435-442.
Mahati K, Bhagya V, Christofer T, Sneha A, Shankaranarayana Rao BS. Enriched environment ameliorates depression-induced cognitive deficits and restores abnormal hippocampal synaptic plasticity. Neurobiol Learn Mem 2016;134:379-391.
Mohanraj K, Karthikeyan BS, Vivek-Ananth RP, Chand RPB, Aparna SR, et al. IMPPAT: A curated database of Indian medicinal plants, phytochemistry and therapeutics. Sci Rep 2018;8:4329.
Vivek-Ananth RP, Mohanraj K, Sahoo AK, Samal A. IMPPAT 2.0: an enhanced and expanded phytochemical atlas of indian medicinal plants. ACS Omega 2023;8:8827-8845.
Sharma R, Bala E. Network pharmacology and molecular docking approach to elucidate potential phytocompounds, targets, and mechanisms of Glycyrrhizaglabra in the alleviation of cellular senescence. Phytomedicine Plus 2024;4:100579.
Tiwari P, Phadte S, Chandavarkar S, Biradar B, Desai SM. Design, synthesis and characterization of a series of 6-substituted-4-hydroxy-1-(2-substitutedthiazol-4-yl) quinolin-2 (1H)-one derivatives and evaluation of their in vitro anticancer and antibacterial activity. J Indian Chemical Soc 2023;100:100951.
Mohammadi-Farani A, Nazari S, Mohammadi M, Navid SJ, Hosseini A, et al. Novel acetylcholinesterase inhibitors: Synthesis, docking and inhibitory activity evaluation of 4-benzamido-N-(1-benzylpiperidin-4-yl) benzamide derivatives. Results Chem 2024;7:101273.
Mahmudov I, Demir Y, Sert Y, Abdullayev Y, Sujayev A, et al. Synthesis and inhibition profiles of N-benzyl-and N-allyl aniline derivatives against carbonic anhydrase and acetylcholinesterase–A molecular docking study. Arab J Chem 2022;15:103645.
Robinson RC, Radziejewski C, Spraggon G, Greenwald J, Kostura MR, et al. The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site. Protein Science 1999;8:2589-2597.
Furukawa H. Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. EMBO J 2003;22:2873-2885.
Omotuyi OI, Ueda H. Molecular dynamics study-based mechanism of nefiracetam-induced NMDA receptor potentiation. Comput Biol Chem 2015;55:14-22.
Eronen V, Tullila A, Iljin K, Rouvinen J, Nevanen TK, et al. Structural insight to elucidate the binding specificity of the anti-cortisol Fab fragment with glucocorticoids. J Struct Biol 2023;215: 107966.
Rahmani AH, Almatroudi A, Alrumaihi F, Khan AA. Pharmacological and therapeutic potential of neem (Azadirachtaindica). Phcog Rev 2018;12:250-255.
Islas JF, Acosta E, Zuca G, Delgado-Gallegos JL, Moreno-Treviño MG, et al. An overview of Neem (Azadirachtaindica) and its potential impact on health. J Functional Foods 2020;74:104171
Sandhir R, Khurana M, Singhal NK. Potential benefits of phytochemicals from Azadirachtaindica against neurological disorders. Neurochem Int 2021;146: 105023.
Ugoeze KC, Oluigbo KE, Chinko BC. Phytomedicinal and Nutraceutical Benefits of the GC-FID Quantified Phytocomponents of the Aqueous Extract of Azadirachtaindica leaves. J Pharm Pharmacol Res 2020;4:149-163.
Hadipour M, Refahi S, Jangravi Z, Meftahi GH. Tarooneh extract relieves anxiety-like behaviors and cognitive deficits by inhibiting synaptic loss in the hippocampus and frontal cortex in rats subjected to chronic restraint stress. Biotech 2023;13:156.
Srikumar BN, Raju TR, Shankaranarayana Rao BS. The involvement of cholinergic and noradrenergic systems in behavioral recovery following oxotremorine treatment to chronically stressed rats. Neuroscience 2006;143:679-688.
Ramkumar K, Srikumar BN, Shankaranarayana Rao BS, Raju TR. Self-stimulation rewarding experience restores stress-induced CA3 dendritic atrophy, spatial memory deficits and alterations in the levels of neurotransmitters in the hippocampus. Neurochem Res 2008;33:1651-1662.
Veena J, Srikumar BN, Mahati K, Bhagya V, Raju TR, et al. Enriched environment restores hippocampal cell proliferation and ameliorates cognitive deficits in chronically stressed rats. J Neurosci Res 2009a;87:831-843.
Christian KM, Miracle AD, Wellman CL, Nakazawa K. Chronic stress-induced hippocampal dendritic retraction requires CA3 NMDA receptors. Neuroscience. 2011; 174: 26-36. doi: 10.1016/j.neuroscience.2010.11.033.
Tripathi SJ, Chakraborty S, Srikumar BN, Raju TR, Shankaranarayana Rao BS. Inactivation of basolateral amygdala prevents chronic immobilization stress-induced memory impairment and associated changes in corticosterone levels. Neurobiol Learn Mem 2017;142:218-229.
Tripathi SJ, Chakraborty S, Srikumar BN, Raju TR, Shankaranarayana Rao BS. Basolateral amygdalar inactivation blocks chronic stress-induced lamina-specific reduction in prefrontal cortex volume and associated anxiety-like behavior. Prog Neuropsychopharmacol Biol Psychiatry 2019; 88:194-207.
Kandhare AD, Mukherjee AA, Bodhankar SL. Neuroprotective effect of Azadirachtaindica standardized extract in partial sciatic nerve injury in rats: Evidence from anti-inflammatory, antioxidant and anti-apoptotic studies. EXCLI J 2017;16:546-565.
Xiang X, Wu L, Mao L, Liu Y. Anti‑oxidative and anti‑apoptotic neuroprotective effects of Azadirachtaindica in Parkinson‑induced functional damage. Mol Med Rep 2018;17:7959-7965.
Raghavendra M, Maiti R, Kumar S, Acharya S. Role of aqueous extract of Azadirachtaindica leaves in an experimental model of Alzheimer's disease in rats. Int J Appl Basic Med Res 2013;3: 37-47.
| Files | ||
| Issue | Vol 10, No 3, 2025 | |
| Section | Research Article(s) | |
| DOI | https://doi.org/10.18502/tim.v10i3.19758 | |
| Keywords | ||
| Azadirachta indica Neem Chronic immobilization stress Memory impairment Acetylcholinesterase Molecular docking Behavioral tests Neuroprotection | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
